a) Field of the Invention
The present invention relates to proteins which inhibit the activation of transcription factors, particularly of the transcription factor NF.kappa.B. The invention is also directed to the recombinant production of these proteins, particularly in vivo, and to methods for treating adult respiratory distress syndrome (ARDS), asthma, allograft rejection, vasculitis, and vascular restenosis, by means of genetic therapy, as well as other conditions that are typically responsive to inhibition of NF.kappa.B.
b) Description of Related Art
During inflammation, the expression of a number of different genes is up-regulated in epithelial and endothelial cells, including those coding for interleukins, transcription factors, adhesion molecules, and components of the coagulation system. Transcription of many of these genes involves the transcription factor NF.kappa.B.
The transcription factor NF.kappa.B is constitutively expressed in the cytoplasm of cells. Induction of gene transcription by NF.kappa.B-like proteins results from post-translational modification permitting translocation of the preformed transcription factor from the cytoplasm to the nucleus. This translocation is controlled by the phosphorylation and degradation of an inhibitor protein called I.kappa.B, which forms a complex with NF.kappa.B, and thereby holds it in the cytoplasm. Stimulation of the cell by appropriate signals leads to modification of I.kappa.B which in turn results in its dissociation from NF.kappa.B.
Binding of the I.kappa.B protein to NF.kappa.B masks the nuclear localization signal (NLS) of NF.kappa.B. Upon stimulation of the cell with specific agents, which depend on the cell type and stage of cell development, I.kappa.B is modified in a way that disables binding to NF.kappa.B, leading to dissociation of NF.kappa.B from I.kappa.B. Signals leading to this modification are believed to involve the generation of oxygen radicals, or kinase activation, and to lead to phosphorylation of I.kappa.B at specific sites; particularly at .sup.32 Ser, .sup.36 Ser, and .sup.42 Tyr. As a result its NLS is unmasked and NF.kappa.B is translocated to the nucleus, where it binds to specific DNA sequences in the regions which control gene expression. NF.kappa.B binding to these sites leads to transcription of genes involved in the inflammatory process.
The transcription factor NF.kappa.B was originally isolated from mature B cells where it binds to a decameric sequence motif in the .kappa. light chain enhancer. Although NF.kappa.B was initially believed to be specific for this cell type and this stage of cell development, NF.kappa.B-like proteins have since been identified in a large number of cell types and, as discussed above, have been shown to be more generally involved in the induction of gene transcription. This has been further supported by the identification of functionally active NF.kappa.B binding sites in several inducible genes.
NF.kappa.B is a heterodimeric protein consisting of a 50 kD subunit (p50) and a 65 kD subunit (p65). The cDNAs for p50 and p65 have been cloned and have been shown to be homologous over a region of 300 amino acids. The p50 subunit shows significant homology to the products of the c-rel protooncogene isolated from mammals and birds, and to the Drosophila gene product of dorsal. Recently an additional member of the NF.kappa.B family, relB, has been cloned as an immediate early response gene from serum-stimulated fibroblasts.
Both p50 and p65 are capable of forming homodimers, although with different properties: whereas p50 homodimers have strong DNA binding affinity but cannot transactivate transcription, the p65 homodimers can only weakly bind to DNA but are capable of transactivation. p50 is synthesized as the amino-terminal part of the 110 kD precursor (p1110), which has no DNA binding and dimerisation activity. The carboxy-terminal part contains eight ankyrin repeats, a motif found in several proteins involved in cell cycle control and differentiation. Cloning of a shorter (2.6kb) RNA species which is induced in parallel with the 4 kb p50 precursor RNA has revealed that, either by alternative splicing or by differential promoter usage, the C-terminal part of the 110 kD protein can also be expressed independently.
Five I.kappa.b family members have been identified: I.kappa.B-.alpha., I.kappa.B-.beta., p105/I.kappa.B-.gamma., p100/I.kappa.B-.DELTA., and I.kappa.B-.epsilon. (Baeuerle and Baltimore, Cell 87:13-20, 1996). All I.kappa.B-like family members contain multiple ankyrin repeats, which are essential for inhibition of NF-.kappa.B activation.
The I.kappa.B.alpha.-like proteins contain five ankyrin repeats. RL/IF-1 has been cloned and shown to be expressed in regenerating liver within 30 minutes after hepatectomy. Deletion mutagenesis studies have revealed that four out of the five ankyrin repeats of pp40 are essential to inhibit DNA binding activity and to associate with c-rel, and that also the C-terminal region is required. Studies with monospecific antibodies, conducted with the 110 kD p50 precursor, have demonstrated that the C-terminal part (the part with I.kappa.B activity) masks the nuclear localization signal (NLS) located in the amino-terminal region of p50. Brown et al. in Science 267:1485-1488 (1995) reported an I.kappa.B deletion mutant, lacking 54 NH.sub.2 -terminal amino acids, which was neither proteolyzed nor phosphorylated by signals and continued to fully inhibit NF.kappa.B. Scheinman et al. and Auphan et al. have reported that glucocorticoid induced immunosuppression is mediated through induction of I.kappa.B synthesis (Science, 270:283-285 and 286-290 (1995)).
It is an object of this invention to provide I.kappa.B-like muteins which are not deactivated in vivo and thus continue to inhibit NF.kappa.B and impede or prevent the induction of inflammation.